Guest Column | May 31, 2023

How Predictive Can Regulatory Drug Abuse Potential Investigations Be in Animals?

By Anke Rosch, Boehringer Ingelheim


Is addiction attributed to “the animal or the human within us”1 or is substance abuse unique to humans? This question has intrigued me since the EMA guidance on Non-clinical Investigation Of The Dependence Potential Of Medicinal Products in 20062 and the U.S. FDA guidance for industry on the Assessment of Abuse Potential of Drugs in 20103 (draft) have requested the performance of dedicated studies in animals.

Regulatory Considerations

Both the EMA and FDA guidance made investigations mandatory on the abuse potential for CNS active drugs that are novel or that have a chemical structure and/or mechanism of action similar to known substances of abuse and for their major metabolites.2,3 Part 1 of this article series4 detailed early preclinical investigations enabling the assignment of drug candidates to this class of concern.

For preclinical abuse potential testing in animals, EMA2 defined first tier tests as primary and secondary pharmacodynamic (safety pharmacology) studies.5 These tests investigate CNS effects of a substance either related or unrelated to its therapeutic target, respectively, at therapeutic doses and above. As second tier tests, EMA2 identified preclinical in-vivo abuse potential studies. For simplicity, the FDA guidance3 summarized all preclinical in-vivo work under the term “animal abuse-related studies.”

First Tier Tests

As part of the core battery in safety pharmacology,5 the modified Irwin test6 or the functional observation battery7 in mice and rats is performed before Phase 1 clinical studies. They serve for general investigation of behavioral, neurological, and locomotor activity effects after drug treatment at therapeutic up to toxic doses. Effects are compared mostly to a vehicle but not to a positive control group, which is mandatory for preclinical abuse potential studies. These tests enable the separation of sedative or stimulant properties of a drug8 and the identification of abuse–related clinical signs such as head twitches or wet dog shakes.9 The results may support the choice of a positive control2, 3 for dedicated preclinical abuse potential studies, especially for drugs with a new mechanism of action (MoA).

Positive signals in preclinical locomotor activity investigations in rats and mice, such as decreases (depressant, e.g., morphine) or increases (stimulant, e.g., amphetamine), are a flag for further dedicated abuse potential studies. They need to be separated in their classification from anxiogenic,10 sedative, and anorectic effects or from a drug-induced disruption of working memory.11 Increased sensation-seeking in humans that preclinically translates to an increased spontaneous locomotor activity in a novel environment12 raises the vulnerability to addiction13 to substances such as cocaine14 and nicotine.15 Rats with a higher locomotor activity achieved higher levels of self-administration of, e.g., cocaine16 and amphetamine17 but not morphine.18 This inconsistency leaves the question open whether increased locomotor activity is predictive for a higher propensity to self-administer drugs19 or for differences in learning an operant task.20

Several publications have challenged the relevance of preclinical CNS findings for prediction of human adverse drug reactions. Olson and colleagues21 showed about 60% concordance based on 150 drugs in 2000, in contrast with Tamaki et al.22 with only 26% correlation based on 142 drugs in 2013. Meade et al. (2015)23 concluded that the rodent Irwin test/functional observation battery neither predicts nor detects most common adverse events in human Phase 1 (141 drugs). 

How much of human addiction is shared with animals and can be modeled successfully?

Second Tier Tests

Literature presents addiction-like behavior in the wild, such as elephants enjoying the fermented fruits of the Marula tree in Africa,24 wallabies getting high on poppy plants,25 and big-horned sheep with preference for hallucinogenic lichen,26 thus supporting modeling in animals. 

Dedicated tests requested by EMA2 and FDA,3 such as drug discrimination and intravenous self-administration (IVSA), are usually performed in non-standard rat strains after Phase 2 clinical studies. The selected dose range ideally results in a two- to three-fold multiple of the maximum human therapeutic plasma concentration (FDA)3 or is pushed up to the maximum tolerated dose (EMA)2 as long as the animal can successfully pass the test.

In the drug discrimination paradigm in a two-lever operant chamber, rats are trained with a known agent of abuse to discriminate “drug” (training drug-like) from “non-drug” (e.g., vehicle-like) by pressing one of two different levers. Various administration routes are feasible. The similarity between the new drug and the training drug is graded as full generalization (>80% drug appropriate lever pressing), partial generalization (60%-80%), or no generalization (<20 %).3

Predictive testing highly depends on the choice of a pharmacologically “similar” training drug.27,28 For example, rats trained with the µ-opioid agonist fentanyl showed generalization to other µ-opioid agonists but not to haloperidol, a dopamine D2 antagonist.29 Impressive test selectivity within a receptor system was proven with GABA modulating substances. Baboons trained with lorazepam showed generalization to other benzodiazepines, only partial generalization to barbiturates, and no generalization to the minor tranquillizer meprobamate.30, 31, 32 This raises doubts whether we can prove generalization at all when a drug with a new MoA shows only a marginal neuropharmacological overlap with the training drug and raises the question how we should interpret partial generalization.

The training and positive control drugs for compounds with a new MoA are often selected in relation to their target or off-target activity, or animals are tried to be trained by the drug candidate. Ator and Griffith33 characterized the use of a training drug and positive control based on the similarity of effects of the drug candidate on general condition in animals like sedation or stimulation as subordinate.

Horton and colleagues34 showed in a literature search an overall concordance of 69% between the drug discrimination results and the Controlled Substances Act (CSA)35 scheduling status based on 100 drugs.

The investigation of rewarding properties of new drugs, e.g., by the intravenous self-administration paradigm, is less dependent on the choice of the training drug.

The self-administration paradigm as a form of an operant conditioning investigates whether a new drug produces a similar pleasurable psychoactive experience, like a known drug of abuse. After training with food rewards and a reinforcing scheduled training drug, rats equipped with chronic indwelling jugular cannula may repeatedly start self-administration of the compound by lever pressing.

Because of the frequent failure of drugs developed for substance use disorder,36 this test scenario was caught in the crossfire, starting with the “Rat Park Project”37 in 1970. A series of studies38,39 challenged whether the more frequent decision of isolated rats to self-administer morphine compared to socially housed rats indicated the rewarding properties of the drug or a lack of choice, now known to be shared with human drug addicts.40,41 Investigations by Ahmed and colleagues supported the conclusion that we cannot model human addiction, showing that most rats (85%-90%) stopped their escalating drug intake when they had free access to an alternative reinforcer such as saccharin.42,43 Based on historical evaluations of individuals over five years, a small minority of 8.7% of rats44 expressed a continuous preference for intravenous cocaine. The number of human cocaine users resulting in less than 10% becoming addicted45 shows surprising similarities with the situation in animals.

There is proof, however, that nearly all drugs abused in humans could be self-administered46, 47 in animals. O’Connor and colleagues showed in literature concordant findings in the rat self-administration study with at least one clinical abuse indicator in 64 (90.1%) of 71 drugs.48 Fixed ratio or progressive ratio IVSA studies could predict the later scheduling status in 78% or 71% of cases, respectively.34 In contrast, a poster presented by FDA representatives in 201949 reviewed the outcome of preclinical abuse potential studies between 2009 and 2019 for eight molecular entities with a new MoA supervised in the CSA.35 Five out of six drugs tested in the IVSA paradigm and four out of eight monitored in the drug discrimination assay in rats and/or monkeys showed negative results in contrast to clinical studies.


The article describes a selection of animal models for preclinical abuse potential testing that are mentioned in EMA2 and FDA3 guidelines.

Numerous publications have challenged the validity of preclinical models for the development of new medications. This does not surprise. The multifaceted nature of this disorder in humans as a neurochemical brain disease and a disorder of choice affected by environmental factors50 is hard to model in animals.

For preclinical abuse potential testing, the predictivity is considered acceptable based on retrospectively generated, concordant results for substances known to be addictive. When a preclinical decision about the abuse potential of a compound needs to be done prospectively, the lack of appropriate positive control and training substances, equivocal findings such as partial generalization, and the variability among the study animals may aggravate classification. Extended testing with more than the standard preclinical models, including more than one training /positive control drug for substances with a new MoA, collides with the goals of speeding up the development of new medications and of reducing the number of animals.

Horton and colleagues34 dampened the enthusiastic hope for unequivocal results of human abuse potential studies. They showed that combinations of, e.g., preclinical with clinical studies have a similar predictivity for the scheduling status as one of the individual studies alone. I am excited to see further progress of these testing strategies within the next decade in relation to the conceptualization of substance use disorder as unique to humans or successfully testable in animal models.


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About The Author:

Anke Rosch is a board-certified pharmacologist and toxicologist working at Boehringer Ingelheim Pharma GmbH & Co. KG. A doctor of veterinary medicine, she has more than 20 years of experience in the pharmaceutical industry and has special expertise in safety pharmacology. Anke can be reached at